?DESCRIPTION (provided by applicant): Intercellular communication by the hedgehog cell signaling pathway is known to be essential for human craniofacial and dental development, and the ligand protein Sonic hedgehog (Shh) has been established as a key molecule during odontogenesis. However, it is incompletely understood exactly how Shh controls the cellular mechanisms of odontogenesis and which other cell signaling pathways influence this control. The long- term goal of this research is to understand how cell signaling instructs embryonic tissues to adopt the correct morphology and organization to produce a mature tooth of the proper shape and size. The objectives of this proposal are to learn more precisely the developmental cellular phenotypes that Shh signaling controls during tooth formation and how other signaling pathways may be required for this control, by exploiting advances in genomics and imaging technologies using the zebrafish vertebrate model system. The central hypothesis guiding this proposal is that secretion of Shh from the inner dental epithelium regulates tooth size and shape by directing the movement and proliferation of cranial neural crest cells during the formation of the dental papilla, and that signaling from other pathways (EDA, FGF, and/or RA) modulates this hedgehog function. The rationale for the proposed work is that a more exact understanding of the cellular behaviors that hedgehog signaling controls during odontogenesis will provide the mechanistic information needed to target the hedgehog pathway in translational dental therapies, such as regrowing teeth de novo in humans, and maximize the chance of their success. Guided by strong preliminary data, the hypothesis will be tested by completing the following two specific aims: 1) manipulate hedgehog activity in vivo to test the role of Shh in the formation of the dental papilla and 2) Test interactions between hedgehog and other signaling pathways during odontogenesis. Under the first aim, the approach will be to spatiotemporally modulate levels of Shh activity and observe the consequent effects within tooth germs on gene expression, cell movement, cell proliferation, and tooth morphogenesis. Under the second aim, the approach will be to simultaneously manipulate levels of hedgehog signaling and those of other pathways known to be able to functionally alter zebrafish tooth development including EDA, FGF, and RA, looking for additive or synergistic effects. Preliminary data indicate that Shh may play a previously unknown, specific, and essential role in the formation of the dental papilla. This proposed research is innovative; because it will exploit the evolutionary position of zebrafish as a vertebrate as well as the experimental accessibility of this model system to quickly obtain data that would be more time consuming, costly, or even impossible to obtain in mammals. The contribution of the proposed work will be significant because it will provide new information about how hedgehog signaling specifically directs cells during their participation in tooth formation and may allow for more directed targeting of the hedgehog pathway and the cells that it controls in translational dental therapies such as human tooth regeneration.